Semaphorin polypeptides

ABSTRACT

The invention is directed to semaphorin polypeptide as a purified and isolated protein, the DNA encoding the semaphorin polypeptide, host cells transfected with cDNAs encoding the polypeptide and methods for preparing the semaphorin polypeptide.

FIELD OF THE INVENTION

[0001] The present invention relates to semaphorin polypeptides, nucleicacids encoding such semaphorin polypeptides, processes for producingrecombinant semaphorin polypeptides, pharmaceutical compositionscontaining such polypeptides relates and processes for treatingdisorders associated with semaphorin activity.

BACKGROUND OF THE INVENTION

[0002] The semaphorin gene family includes a large number of moleculesthat encode related transmembrane and secreted glycoproteins known to beneurologic regulators. The semaphorins are generally well conserved intheir extracellular domains which are typically about 500 amino acids inlength. Semaphorin family proteins have been observed in neuronal andnonneuronal tissue and have been studied largely for their role inneuronal growth cone guidance. For example, the secreted semaphorinsknown as collapsin-1 and Drosophila semaphorin II are selectivelyinvolved in repulsive growth cone guidance during development. Flieshaving semaphorin II genes that are mutated so that their function isreduced exhibit abnormal behavior characteristics.

[0003] Another semaphorin gene has been identified in several strains ofpoxvirus. This semaphorin is found in vaccinia virus (Copenhagen strain)and Ectromelia virus, and is encoded in an open reading frame (ORF)known as A39R. The A39R encoded protein has no transmembrane domain andno potential membrane linkage and is known to be a secreted protein.Variola virus ORF also contains sequences that share homology with thevaccinia virus ORF A39R at the nucleotide level and the amino acidlevel. Another viral semaphorin. AHV-sema, has been found in theAlcelaphine Herpesvirus (AHV).

[0004] Genes encoding mammalian (human, rat, and mouse) semaphorins havebeen identified, based upon their similarity to insect semaphorins.Functional studies of these semaphorins suggest that embryonic and adultneurons require a semaphorin to establish workable connections.Significantly, the fast response time of growth cone cultures toappropriate semaphorins suggests that semaphorin signaling involves areceptor-mediated signal transduction mechanism. Semaphorin ligands thatare secreted into the extracellular milieu signal through receptorbearing cells in a located and systemic fashion. In order to furtherinvestigate the nature of cellular processes regulated by such local andsystemic signaling, it would be beneficial to identify additionalsemaphorin receptors and ligands. Furthermore, because virus encodedsemaphorins are produced by infected cells and are present in virusesthat are lytic (poxviruses) and viruses that are not known to beneurotropic (AHV), it is unlikely that their primary function is tomodify neurologic responses. It is more likely that the virus encodedsemaphorins function to modify the immunologic response of the infectedhost and it is likely that mammalian homologues to virus encodedsemaphorins function to modify the immunologic response. In view of thesuggestion that viral semaphorins may function in the immune system asnatural immunoregulators it would be beneficial to identify semaphorinsthat may be therapeutic agents for enhancing or diminishing the immuneresponse.

SUMMARY OF THE INVENTION

[0005] The present invention pertains to novel semaphorins as isolatedor homogeneous proteins. In particular, the present invention providessemaphorin polypeptides, that are homologous to the viral semaphorinsA39R and AHVSema. Within the scope of the present invention are DNAsencoding semaphorin polypeptides, and expression vectors that includeDNA encoding semaphorin polypeptides. The present invention alsoincludes host cells that have been transfected or transformed withexpression vectors that include DNA encoding a semaphorin polypeptide,and processes for producing semaphorin polypeptides by culturing suchhost cells under conditions conducive to expressing semaphorinpolypeptides. The present invention further includes antibodies directedagainst semaphorin polypeptides.

[0006] Further within the scope of the present invention are processesfor purifying or separating certain novel semaphorin polypeptides orcells that express novel semaphorins to which semaphorin receptorpolypeptides bind. Such processes include binding at least onesemaphorin receptor to a solid phase matrix and contacting a mixturecontaining a semaphorin polypeptide to which the semaphorin receptorbinds, or a mixture of cells expressing the semaphorin with the boundsemaphorin receptor, and then separating the contacting surface and thesolution.

[0007] The present invention additionally provides processes fortreating mammals afflicted with a disease that is ameliorated by theinteraction of semaphorins and their receptors. Such processes involveactivating immune cells that express receptors for the semaphorins ofthe present invention and include administering a therapeuticallyeffective amount of semaphorin to a mammal afflicted with the disease.The therapeutically effective amount is sufficient to activate immunesystem cells that express semaphorin receptors. Such an activationresults in the secretion of cytokines, the regulation of activationantigens or the migration of the cell to sites of immune activity.

DETAILED DESCRIPTION OF THE INVENTION

[0008] The present invention provides novel semaphorin polypeptides, DNAencoding semaphorin polypeptides and recombinant expression vectors thatinclude DNA encoding semaphorin polypeptides. The present inventionfurther provides methods for isolating semaphorin polypeptides andmethods for producing recombinant semaphorin polypeptides by cultivatinghost cells transfected with the recombinant expression vectors underconditions appropriate for expressing semaphorin and recovering theexpressed semaphorin polypeptide.

[0009] This invention additionally provides antibodies directed againstsemaphorin polypeptides.

[0010] Particular semaphorin embodiments of the present inventioninclude polypeptides homologous to AHVsema. The native semaphorinpolypeptide described herein was discovered using data base search andcomparison techniques that resulted in the identification of at leastone EST having some homology to viral semaphorins. As described inExample 1, PCR techniques were used to identify and clone the full viralsemaphorin homologue. The human semaphorin of the present invention isfound in placenta, testis, ovary, spleen, dendritic cells, and B cells.Semaphorin polypeptides of the present invention bind to the semaphorinreceptor, designated VESPR (described in copending application S/N08/958,598, incorporated herein by reference). Evidence suggests thatthe interaction between the semaphorins of the present invention andtheir receptors are associated with the immune suppression of maturedendritic cells. For this reason, the semaphorins of the presentinvention are designated DCSema.

[0011] Example 1 describes identifying a native DCSema of the presentinvention. The amino acid sequence of the identified native DCSema isdisclosed in SEQ ID NO:2 and the DNA encoding the amino acid sequence isdisclosed in SEQ ID NO:1. The amino acid sequence presented in SEQ IDNO:2 is a secreted soluble polypeptide, but may additionally exist as amembrane bound protein. The amino acid sequence of SEQ ID NO:2 has apredicted signal sequence that includes amino acids 1-44. Alsoencompassed within the present invention are soluble DCSema polypeptidesthat lack the signal sequence. An example of such a polypeptide is aminoacids 45-666 of SEQ ID NO:2. The EST 151129 portion of SEQ ID NO:2 has a28% identity to A39R and 44% identity to AHVsema, both of which bind toVESPR, a semaphorin receptor described in copending patent applicationS/N 08/958,598. A39R and AHVsema share 29% identity.

[0012] The terms “semaphorin polypeptide”, “human semaphorin homologue”,“DCSema” and homologues of AHVSema encompass polypeptides having theamino acid sequence disclosed in SEQ ID NO:2, and proteins that areencoded by nucleic acids that contain the nucleic acid sequence of SEQID NO: 1. In addition, those polypeptides that have a high degree ofsimilarity or a high degree of identity with the amino acid sequence ofSEQ ID NO:2, which polypeptides are biologically active and bind atleast one molecule or fragments of a molecule that is a semaphorinreceptor. In addition, “semaphorin polypeptide”, “human semaphorinhomologue”, “DCSema” refers to biologically active gene products of theDNA of SEQ ID NO: 1. Further encompassed by semaphorin polypeptides aresoluble or truncated proteins that comprise primarily the bindingportion of the protein, retain biological activity and are capable ofbeing secreted. Specific examples of such soluble proteins are thosecomprising the sequence of amino acids 45-666 of SEQ ID NO:2.

[0013] The term “biologically active” as it refers to semaphorinpolypeptides, means that the semaphorin polypeptide is capable ofbinding to at least one semaphorin receptor. Assays suitable fordetermining DCSema binding are described infra and can include standardflow cytometry tests and slide binding tests.

[0014] “Isolated” means a DCSema polypeptide is free of association withother proteins or polypeptides, for example, as a purification productof recombinant host cell culture or as a purified extract.

[0015] A DCSema polypeptide variant as referred to herein, means apolypeptide substantially homologous to native DCSema polypeptide, butwhich has an amino acid sequence different from that of the nativepolypeptides because of one or more deletions, insertions orsubstitutions. The variant amino acid sequence preferably is at least80% identical to a native DCSema amino acid sequence, most preferably atleast 90% identical. The percent identity may be determined, forexample, by comparing sequence information using the GAP computerprogram, version 6.0 described by Devereux et al. (Nucl. Acids Res.12:387, 1984) and available from the University of Wisconsin GeneticsComputer Group (UWGCG). The preferred default parameters for the GAPprogram include: (1) a unary comparison matrix (containing a value of 1for identities and 0 for non-identities) for nucleotides, and theweighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endcaps. Variants may comprise conservatively substituted sequences,meaning that a given amino acid residue is replaced by a residue havingsimilar physiochemical characteristics. Examples of conservativesubstitutions include substitution of one aliphatic residue for another,such as Ile, Val, Leu, or Ala for one another, or substitutions of onepolar residue for another, such as between Lys and Arg; Glu and Asp; orGin and Asn. Other such conservative substitutions, for example,substitutions of entire regions having similar hydrophobicitycharacteristics, are well known. Naturally occurring variants or allelesare also encompassed by the invention. Examples of such variants areproteins that result from alternate mRNA splicing events or fromproteolytic cleavage of the DCSema protein, wherein the binding propertyis retained. Alternate splicing of mRNA may yield a truncated butbiologically active polypeptide, such as a naturally occurring solubleform of the protein, for example. Variations attributable to proteolysisinclude, for example, differences in the N-termini upon expression indifferent types of host cells, due to proteolytic removal of one or moreterminal amino acids from the DCSema polypeptide (generally from 1-5terminal amino acids).

[0016] Example 3 describes the construction of a novel viralSemaphorin/Fc fusion (DCSema/Fc) protein that may be utilized instudying the biological characteristics of DCSema and their receptordistribution. Other antibody Fc regions may be substituted for the humanIgG1 Fc region described in Example 3. Suitable Fc regions are thosethat can bind with high affinity to protein A or protein G, and includethe Fc region of human IgG1 or fragments of the human or murine IgG1 Fcregion, e.g., fragments comprising at least the hinge region so thatinterchain disulfide bonds will form. The viral DCSema/Fc fusion proteinoffers the advantage of being easily purified. In addition, disulfidebonds form between the Fc regions of two separate fusion protein chains,creating dimers.

[0017] The soluble DCSema polypeptides of the present invention may beisolated and identified by separating intact cells that express thedesired protein from the culture medium in which the cells grow, andthen assaying the medium (supernatant) for the presence of the desiredprotein. Separation can be accomplished using standard separationtechniques including, including centrifugation. The presence of theDCSema polypeptide in the medium indicates that the protein was secretedfrom the cells in its expected soluble form. Because the DCSemapolypeptides of the present invention are secreted soluble polypeptidesthey possess many advantages over membrane bound proteins. Purificationof the proteins from recombinant host cells is feasible, since thesoluble proteins are secreted from the cells. Further, soluble proteinsare generally more suitable for intravenous administration.

[0018] Truncated DCSema proteins comprising less than the entiresecreted polypeptide are included in the invention, e.g. solublefragments such as amino acids 52-543 of SEQ ID NO:2 that include the“semaphorin domain,” which is part of an active binding site, and aminoacids 45-644 of SEQ ID NO:2 that include the secreted protein withoutthe signal peptide, are included in the invention. When initiallyexpressed within a host cell, DCSema polypeptides may additionallycomprise one of the heterologous signal peptides described below that isfunctional within the host cells employed. Alternatively, the proteinmay comprise the native signal peptide. In one embodiment of theinvention, DCSema polypeptides can be expressed as a fusion proteincomprising (from N— to C-terminus) the yeast α-factor signal peptide, aFLAG® peptide described below and in U.S. Pat. No. 5,011,912, andsoluble DCSema polypeptide consisting of amino acids 45 to 666 of SEQ IDNO:2. This recombinant fusion protein is expressed in and secreted fromyeast cells. The FLAG® peptide facilitates purification of the protein,and subsequently may be cleaved from the DCSema using bovine mucosalenterokinase.

[0019] Truncated DCSema polypeptides may be prepared by any of a numberof conventional techniques. A desired DNA sequence may be chemicallysynthesized using techniques known per se. DNA fragments also may beproduced by restriction endonuclease digestion of a full length clonedDNA sequence, and isolated by electrophoresis on agarose gels. Linkerscontaining restriction endonuclease cleavage site(s) may be employed toinsert the desired DNA fragment into an expression vector, or thefragment may be digested at cleavage sites naturally present therein.The well known polymerase chain reaction procedure also may be employedto amplify a DNA sequence encoding a desired protein fragment. As afurther alternative, known mutagenesis techniques may be employed toinsert a stop codon at a desired point, e.g., immediately downstream ofthe codon for the last amino acid of the binding domain.

[0020] As stated above, the invention provides isolated or homogeneousDCSema polypeptides, both recombinant and non-recombinant. Variants andderivatives of native DCSema proteins that retain the desired biologicalactivity (e.g., the ability to bind to DCSema receptors) may be obtainedby mutations of nucleotide sequences coding for the native polypeptides.Alterations of the native amino acid sequence may be accomplished by anyof a number of conventional methods. Mutations can be introduced atparticular loci by synthesizing oligonucleotides containing a mutantsequence, flanked by restriction sites enabling ligation to fragments ofthe native sequence. Following ligation, the resulting reconstructedsequence encodes an analog having the desired amino acid insertion,substitution, or deletion.

[0021] Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene whereinpredetermined codons can be altered by substitution, deletion orinsertion. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods. Plenum Press, 1981);Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methodsin Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462all of which are incorporated by reference.

[0022] Native DCSema polypeptides may be modified to create DCSemaderivatives by forming covalent or aggregative conjugates with otherchemical moieties, such as glycosyl groups, lipids, phosphate, acetylgroups and the like. Covalent derivatives of the polypeptides may beprepared by linking the chemical moieties to functional groups on DCSemaamino acid side chains or at the N-terminus or C-terminus of a DCSemapolypeptide. Other derivatives of the polypeptides within the scope ofthis invention include covalent or aggregative conjugates of DCSemapolypeptides or their fragments with other proteins or polypeptides,such as by synthesis in recombinant culture as N-terminal or C-terminalfusions. For example, the conjugate may comprise a signal or leaderpolypeptide sequence (e.g. the α-factor leader of Saccharomyces) at theN-terminus of a polypeptide of the present invention. The signal orleader peptide co-translationally or post-translationally directstransfer of the conjugate from its site of synthesis to a site inside oroutside of the cell membrane or cell wall.

[0023] DCSema polypeptide fusion proteins can comprise peptides added tofacilitate purification and identification of semaphorin receptors thatbind semaphorins of the present invention, including VESPR (seecopending application S/N08/958,598. Such peptides include, for example,poly-His or the antigenic identification peptides described in U.S. Pat.No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988.

[0024] The invention further includes DCSema polypeptides with orwithout associated native-pattern glycosylation. DCSema polypeptideexpressed in yeast or mammalian expression systems (e.g., COS-7 cells)may be similar to or significantly different from a native DCSemapolypeptide in molecular weight and glycosylation pattern, dependingupon the choice of expression system. Expression of DCSema polypeptidesin bacterial expression systems, such as E. coli, providesnon-glycosylated molecules.

[0025] Equivalent DNA constructs that encode various additions orsubstitutions of amino acid residues or sequences, or deletions ofterminal or internal residues or sequences not needed for biologicalactivity or binding are encompassed by the invention. For example,N-gycosylation sites in the DCSema extracellular domain can be modifiedto preclude glycosylation, allowing expression of a reduced carbohydrateanalog in mammalian and yeast expression systems. N-glycosylation sitesin eukaryotic polypeptides are characterized by an amino acid tripletAsn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr. Thenative human DC Sema of the present invention comprises 4 such aminoacid triplets, at amino acids 105-107, 157-159, 258-260 and 602-604 ofSEQ ID NO:2. Appropriate substitutions, additions or deletions to thenucleotide sequence encoding these triplets will result in prevention ofattachment of carbohydrate residues at the Asn side chain. Alteration ofa single nucleotide, chosen so that Asn is replaced by a different aminoacid, for example, is sufficient to inactivate an N-glycosylation site.Known procedures for inactivating N-glycosylation sites in proteinsinclude those described in U.S. Pat. No. 5,071,972 and EP 276,846,hereby incorporated by reference.

[0026] In another example, sequences encoding Cys residues that are notessential for biological activity can be altered to cause the Cysresidues to be deleted or replaced with other amino acids, preventingformation of incorrect intramolecular disulfide bridges uponrenaturation. Other equivalents are prepared by modification of adjacentdibasic amino acid residues to enhance expression in yeast systems inwhich KEX2 protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys-Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites. The DCsema of SEQ ID NO:2 contains 7 KEX2 protease processing sites at aminoacids 123-124, 135, 136, 192, 193, 204-205, 460461, 474-475 and 475-476,Nucleic acid sequences within the scope of the invention includeisolated DNA and RNA sequences that hybridize to DCSema nucleotidesequences disclosed herein under conditions of moderate or highstringency, and that encode biologically active DCSema. Conditions ofmoderate stringency, as defined by Sambrook et al. Molecular Cloning: ALaboratory Manual, 2 ed. Vol. 1, pp. 101-104, Cold Spring HarborLaboratory Press, (1989), include use of a prewashing solution of 5×SSC,0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions of about 55°C., 5×SSC, overnight. Conditions of severe stringency include highertemperatures of hybridization and washing. The skilled artisan willrecognize that the temperature and wash solution salt concentration maybe adjusted as necessary according to factors such as the length of thenucleic acid molecule and the relative amount of A, T[U, C and Gnucleotides.

[0027] Due to the known degeneracy of the genetic code wherein more thanone codon can encode the same amino acid, a DNA sequence may vary fromthat shown in SEQ ID NO:1 and still encode a polypeptide having theamino acid sequence of SEQ ID NO:2. Such variant DNA sequences mayresult from silent mutations (e.g., occurring during PCR amplification),or may be the product of deliberate mutagenesis of a native sequence.

[0028] The invention provides equivalent isolated DNA sequences encodingbiologically active human DCSema, selected from: (a) cDNA comprising thenucleotide sequence presented in SEQ ID NO: 1; (b) DNA capable ofhybridization to a DNA of (a) under moderately stringent conditions andthat encodes biologically active polypeptides and (c) DNA that isdegenerate as a result of the genetic code to a DNA defined in (a), or(b) and that encodes biologically active human DCSema polypeptide.Polypeptides encoded by such DNA equivalent sequences are encompassed bythe invention.

[0029] DNAs that are equivalent to the DNA sequence of SEQ ID NO:1include those that hybridize under moderately and highly stringentconditions to the DNA sequence that encodes polypeptides comprising thesequence of SEQ ID NO:2. Examples of proteins encoded by such DNA,include, but are not limited to, polypeptide fragments and proteins thathave inactivated N-glycosylation site(s), inactivated KEX2 proteaseprocessing site(s), or conservative amino acid substitution(s), asdescribed above. DCSema polypeptides encoded by DNA derived from otherspecies, wherein the DNA will hybridize to the cDNA of SEQ ID NO:1, arealso within the present invention.

[0030] DCSema polypeptide variants possessing the ability to bindsemaphorin receptors may be identified by any suitable assay. Biologicalactivity of DCSema polypeptides of the present invention may bedetermined, for example, by competition for binding to the bindingdomain of semaphorin receptors, e.g. competitive binding assays, or forbinding to a semaphorin receptor binding domain.

[0031] One type of a competitive binding assay for a DCSema polypeptideof the present invention uses a radiolabeled DCSema and intactsemaphorin receptor-expressing cells. Instead of intact cells, one couldsubstitute soluble semaphorin receptor:Fc fusion proteins bound to asolid phase through the interaction of a Protein A, Protein G or anantibody to the semaphorin receptor or Fc portions of the molecule, withthe Fc region of the fusion protein. Competitive binding assays can beperformed following conventional methodology. In one embodiment, asoluble semaphorin receptor can be made to compete with an immobilizedreceptor for binding with a soluble semaphorin ligand. For example, aradiolabeled soluble semaphorin ligand can be antagonized by solubleVESPR in an assay for binding activity against a surface-boundsemaphorin receptor. Qualitative results can be obtained by competitiveautoradiographic plate binding assays, or Scatchard plots may beutilized to generate quantitative results.

[0032] Alternatively, semaphorin binding proteins, such as VESPR oranti-semaphorin antibodies, can be bound to a solid phase such as acolumn chromatography matrix or a similar substrate suitable foridentifying, separating or purifying cells that express semaphorin ontheir surface. Binding of a semaphorin-binding protein to a solid phasecontacting surface can be accomplished by any means, for example, byconstructing a VESPR:Fc fusion protein and binding such to the solidphase through the interaction of Protein A or Protein G. Various othermeans for fixing proteins to a solid phase are well known in the art andare suitable for use in the present invention. For example, magneticmicrospheres can be coated with VESPR and held in the incubation vesselthrough a magnetic field. Suspensions of cell mixtures containingsemaphorin-expressing cells are contacted with the solid phase that hasVESPR polypeptides thereon. Cells having semaphorin on their surfacebind to the fixed VESPR and unbound cells then are washed away. Thisaffinity-binding method is useful for purifying, screening or separatingsuch semaphorin-expressing cells from solution. Methods of releasingpositively selected cells from the solid phase are known in the art andencompass, for example, the use of enzymes. Such enzymes are preferablynon-toxic and non-injurious to the cells and are preferably directed tocleaving the cell-surface binding partner. In the case ofsemaphorin-VESPR interactions, the enzyme preferably would cleave thesemaphorin, thereby freeing the resulting cell suspension from the“foreign” semaphorin receptor material. The purified cell populationthen may be used to repopulate mature (adult) tissues.

[0033] Alternatively, mixtures of cells suspected of containingsemaphorin cells first can be incubated with a suitable biotinylatedsemaphorin receptor, e.g. VESPR. Incubation periods are typically atleast one hour in duration to ensure sufficient binding to semaphorinThe resulting mixture then is passed through a column packed withavidin-coated beads, whereby the high affinity of biotin for avidinprovides the binding of the cell to the beads. Use of avidin-coatedbeads is known in the art. See Berenson, et al. J. Cell. Biochem.,10D:239 (1986). Washing unbound material and releasing the bound cellsis performed using conventional methods.

[0034] As described above, cells expressing DCSema polypeptides of thepresent invention can be separated using a semaphorin receptor, e.g.VESPR. In an alternative method, VESPR, other suitable semaphorinreceptors, or an extracellular domain or a fragment thereof can beconjugated to a detectable moiety such as ¹²⁵I to detectsemaphorin-expressing cells. Radiolabeling with ¹²⁵I can be performed byany of several standard methodologies that yield a functional¹²⁵I-molecule labeled to high specific activity or an iodinated orbiotinylated antibody against the semaphorin receptor. Anotherdetectable moiety such as an enzyme that can catalyze a colorimetric orfluorometric reaction, biotin or avidin may be used. Cells to be testedfor DCSema polypeptide-expression can be contacted with a suitablelabeled receptor, e.g. VESPR. After incubation, unbound labeled receptoris removed and binding is measured using the detectable moiety.

[0035] The binding characteristics of DCSema polypeptides may also bedetermined using a conjugated semaphorin receptor (for example,¹²⁵I-semaphorin receptor:Fc) in competition assays similar to thosedescribed above. In this case, however, intact cells expressing DCSemapolypeptide of the present invention, bound to a solid substrate, areused to measure the extent to which a sample containing a putativereceptor variant competes for binding with a conjugated DCSema.

[0036] Other means of assaying for DCSema polypeptides of the presentinvention include the use of anti-DCSema antibodies, cell lines thatproliferate in response to DCSema polypeptide, or recombinant cell linesthat express DCSema and proliferate in the presence of a suitablesemaphorin receptor.

[0037] The DCSema proteins disclosed herein also may be employed tomeasure the biological activity of any semaphorin receptor in terms ofits binding affinity for its semaphorin ligand. As one example, DCSemapolypeptides of the present invention may be used in determining whetherbiological activity is retained after modification of a semaphorinreceptor (e.g., chemical modification, truncation, mutation, etc.). Thebiological activity of a semaphorin receptor thus can be ascertainedbefore it is used in a research study, or in the clinic, for example.

[0038] DCSema polypeptides disclosed herein find use as reagents in“quality assurance” studies, e.g., to monitor shelf life and stabilityof a receptor to which the DCSema binds under different conditions. Toillustrate. DCSema polypeptides of the present invention may be employedin a binding affinity study to measure the biological activity of a testsemaphorin receptor that has been stored at different temperatures, orproduced in different cell types. The binding affinity of the DCSemaprotein for the test receptor is compared to that of a standard orcontrol semaphorin receptor to detect any adverse impact on biologicalactivity of the test semaphorin receptor.

[0039] DCSema polypeptides described herein also find use as carriersfor delivering agents attached thereto to cells expressing semaphorinreceptors to which the semaphorin binds. As described in copendingapplication S/N 958,598, VESPR, to which a DCSema of the presentinvention binds, is expressed in lung epithelial cells, stroma,intestinal epithelial cells and lymphoma cells. DCSema polypeptides ofthe present invention can thus can be used to deliver diagnostic ortherapeutic agents to these cells (or to other cell types found toexpress a suitable semaphorin receptor on cell surfaces) in in vitro orin vivo procedures.

[0040] Diagnostic and therapeutic agents that may be attached to aDCSema polypeptide of the present invention include, but are not limitedto, drugs, toxins, radionuclides, chromophores, enzymes that catalyze acolorimetric or fluorometric reaction, and the like, with the particularagent being chosen according to the intended application. Examples ofdrugs include those used in treating various forms of cancer, e.g.,nitrogen mustards such as L-phenylalanine nitrogen mustard orcyclophosphamide, intercalating agents such ascis-diaminodichloroplatinum, antimetabolites such as 5-fluorouracil,vinca alkaloids such as vincristine, and antibiotics such as bleomycin,doxorubicin, daunorubicin, and derivatives thereof. Among the toxins arericin, abrin, diptheria toxin, Pseudomonas aeruginosa exotoxin A,ribosomal inactivating proteins, mycotoxins such as trichothecenes, andderivatives and fragments (e.g., single chains) thereof. Radionuclidessuitable for diagnostic use include, but are not limited to, ¹²³I, ¹³¹I,^(99m) Tc, ¹¹¹In, and ⁷⁶Br. Radionuclides suitable for therapeutic useinclude, but are not limited to, ¹³¹I, ²¹¹At. ⁷⁷Br, ¹⁸⁶Re, ¹⁸⁸Re, ²¹²Pb,²¹²Bi, ¹⁰⁹Pd, ⁶⁴Cu, and ⁶⁷Cu.

[0041] Such agents may be attached to the DCSema of the presentinvention by any suitable conventional procedure. Semaphorin homologuesof the present invention, being proteins, include functional groups onamino acid side chains that can be reacted with functional groups on adesired agent to form covalent bonds, for example. Alternatively, theprotein or agent may be derivatized to generate or attach a desiredreactive functional group. The derivatization may involve attachment ofone of the bifunctional coupling reagents available for attachingvarious molecules to proteins (Pierce Chemical Company, Rockford, Ill.).A number of techniques for radiolabeling proteins are known.Radionuclide metals may be attached to the receptor by using a suitablebifunctional chelating agent, for example. Conjugates comprisingmolecules of the present invention and a suitable diagnostic ortherapeutic agent (preferably covalently linked) are thus prepared. Theconjugates are administered or otherwise employed in an amountappropriate for the particular application.

[0042] Another use of the DCSema polypeptides of the present inventionis as a research tool for studying the role that the DCSema, inconjunction with semaphorin receptors to which it binds, may play inimmune regulation and viral infection. The polypeptides of the presentinvention also may be employed in in vitro assays for detection ofreceptors to which it binds, e.g. VESPR, or the interactions thereof.Similarly, DCSema polypeptides of the present invention can be used as aresearch tool for studying the role that the ligand, in conjunction withits receptors, play in immune regulation.

[0043] Furthermore, it is known that administration of IL-12 to tumorbearing animals results in tumor regression and the establishment of atumor-specific immune response. Thus, using a DCSema ligand to bind withVESPR in order to enhance or promote IL-12 can induce a curative immuneresponse against aggressive micrometastasizing tumors.

[0044] VESPR, a semaphorin•receptor, binds with a binding partner todown-regulate expression of MHC Class II molecules and CD86, aco-stimulatory molecule, on dendritic cells, cultured with GM-CSF andIL-4 (see copending application S/N 60/085,497). By analogy, thissuggests that the interaction between DCSema of the present inventionand its receptors are associated with the immune suppression of maturedendritic cells. Thus, the use of DCSema ligands, including the DC Semaof the present invention, in the treatment of autoimmune disorders isexpected to diminish unwanted symptoms associated with the autoimmunedisorder by downregulating the antigen presenting capabilities of maturedendritic cells.

[0045] DCSema polypeptides of the invention can be formulated accordingto known methods used to prepare pharmaceutically useful compositions.The molecules of the invention can be combined in admixture, either asthe sole active material or with other known active materials, withpharmaceutically suitable diluents (e.g., Tris-HCl, acetate, phosphate),preservatives (e.g., Thimerosal, benzyl alcohol, parabens), emulsifiers,solubilizers, adjuvants and/or carriers. Suitable carriers and theirformulations are described in Remington's Pharmaceutical Sciences, 16thed. 1980, Mack Publishing Co. In addition, such compositions can containDCSema polypeptide complexed with polyethylene glycol (PEG), metal ions,or incorporated into polymeric compounds such as polyacetic acid,polyglycolic acid, hydrogels, etc., or incorporated into liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts or spheroblasts. Such compositions will influence thephysical state, solubility, stability, rate of in vivo release, and rateof in vivo clearance of DCSema. DCSema polypeptides described herein canbe conjugated to antibodies against tissue-specific receptors, ligandsor antigens, or coupled to ligands of tissue-specific receptors.

[0046] DCSema polypeptides of the present invention can be administeredtopically, parenterally, or by inhalation. The term “parenteral”includes subcutaneous injections, intravenous, intramuscular,intracisternal injection, or infusion techniques. These compositionswill typically contain an effective amount of the polypeptide, alone orin combination with an effective amount of any other active material.Such dosages and desired drug concentrations contained in thecompositions may vary depending upon many factors, including theintended use, patient's body weight and age, and route ofadministration. Preliminary doses can be determined according to animaltests, and the scaling of dosages for human administration can beperformed according to art-accepted practices.

[0047] Polypeptides of the present invention may exist as oligomers,such as covalently-linked or non-covalently-linked dimers or trimers.Oligomers may be linked by disulfide bonds formed between cysteineresidues on different DCSema molecules. In one embodiment of theinvention, a dimer is created by fusing a DCSema to the Fc region of anantibody (e.g., IgG1) in a manner that does not interfere with bindingof the DCSema to a semaphorin receptor-binding domain. The Fcpolypeptide preferably is fused to the C-terminus of a DCSema Generalpreparation of fusion proteins comprising heterologous polypeptidesfused to various portions of antibody-derived polypeptides (includingthe Fc domain) has been described, e.g., by Ashkenazi et al. (PNAS USA88:10535, 1991) and Byrn et al. (Nature 344:677, 1990), herebyincorporated by reference. A gene fusion encoding the DCSema/Fc fusionprotein is inserted into an appropriate expression vector. DCSema/Fcfusion proteins are allowed to assemble much like antibody molecules,whereupon interchain disulfide bonds form between Fc polypeptides,yielding divalent. If fusion proteins are made with both heavy and lightchains of an antibody, it is possible to form a DCSema oligomer with asmany as four semaphorin regions. Alternatively, one can link two DCSemaswith a peptide linker.

[0048] Suitable host cells for expression of DCSema polypeptides of thisinvention include prokaryotes, yeast or higher eukaryotic cells.Appropriate cloning and expression vectors for use with bacterial,fungal, yeast, and mammalian cellular hosts are described, for example,in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, N.Y.,(1985). Cell-free translation systems could also be employed to producethe polypeptides of the present invention using RNAs derived from DNAconstructs disclosed herein.

[0049] Prokaryotes include gram negative or gram positive organisms, forexample, E. coli or Bacilli. Suitable prokaryotic host cells fortransformation include, for example, E. coli, Bacillus subtilis,Salmonella typhimurium, and various other species within the generaPseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic hostcell, such as E. coli, a semaphorin receptor polypeptide may include anN-terminal methionine residue to facilitate expression of therecombinant polypeptide in the prokaryotic host cell. The N-terminal Metmay be cleaved from the expressed recombinant DCSema polypeptide.

[0050] DCSema polypeptides may be expressed in yeast host cells,preferably from the Saccharomyces genus (e.g., S. cerevisiae). Othergenera of yeast, such as Pichia, K. lactis or Kluyveromyces, may also beemployed. Yeast vectors will often contain an origin of replicationsequence from a 2μ yeast plasmid, an autonomously replicating sequence(ARS), a promoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein. 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J.Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900,1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase. 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Hitzeman, EPA-73,657 or in Fleer et.al., Gene, 107:285-195 (1991); and van den Berg et. al., Bio/technology,8:135-139 (1990). Another alternative is the glucose-repressible ADH2promoter described by Russell et al. (J. Biol. Chem. 258:2674, 1982) andBeier et al. (Nature 300:724, 1982). Shuttle vectors replicable in bothyeast and E. coli may be constructed by inserting DNA sequences frompBR322 for selection and replication in E. coli (Amp^(r) gene and originof replication) into the above-described yeast vectors.

[0051] The yeast α-factor leader sequence may be employed to directsecretion of the VESPR or DCSema polypeptide. The α-factor leadersequence is often inserted between the promoter sequence and thestructural gene sequence. See, e.g., Kurjan et al., Cell 30:933, 1982:Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984; U.S. Pat. No.4,546,082; and EP 324,274. Other leader sequences suitable forfacilitating secretion of recombinant polypeptides from yeast hosts areknown to those of skill in the art. A leader sequence may be modifiednear its 3′ end to contain one or more restriction sites. This willfacilitate fusion of the leader sequence to the structural gene.

[0052] Yeast transformation protocols are known to those of skill in theart. One such protocol is described by Hinnen et al., Proc. Natl. Acad.Sci. USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose.10 μg/ml adenine and 20 μg/ml uracil.

[0053] Yeast host cells transformed by vectors containing ADH2 promotersequence may be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Depression of the ADH2 promoter occurs when glucose is exhaustedfrom the medium.

[0054] Mammalian or insect host cell culture systems could also beemployed to express recombinant polypeptides of the present invention.Baculovirus systems for production of heterologous proteins in insectcells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).Established cell lines of mammalian origin also may be employed.Examples of suitable mammalian host cell lines include the COS-7 line ofmonkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981),L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary(CHO) cells. HeLa cells, and BHK (ATCC CRL 10) cell lines, and theCV-1/EBNA-1 cell line derived from the African green monkey kidney cellline CVI (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10: 2821,1991).

[0055] Transcriptional and translational control sequences for mammalianhost cell expression vectors may be excised from viral genomes. Commonlyused promoter sequences and enhancer sequences are derived from Polyomavirus. Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites may be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment which may also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40fragments may also be used, provided the approximately 250 bp sequenceextending from the Hind III site toward the Bgl I site located in theSV40 viral origin of replication site is included.

[0056] Exemplary expression vectors for use in mammalian host cells canbe constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280,1983). A useful system for stable high level expression of mammaliancDNAs in C127 murine mammary epithelial cells can be constructedsubstantially as described by Cosman et al. (Mol. Immunol. 23:935,1986). A useful high expression vector, PMLSV N1/N4, described by Cosmanet al., Nature 312:768, 1984 has been deposited as ATCC 39890.Additional useful mammalian expression vectors are described inEP-A-0367566, and in U.S. patent application Ser. No. 07/701,415, filedMay 16, 1991, incorporated by reference herein. The vectors may bederived from retroviruses. In place of the native signal sequence, andin addition to an initiator methionine, a heterologous signal sequencemay be added, such as the signal sequence for IL-7 described in U.S.Pat. No. 4,965,195; the signal sequence for IL-2 receptor described inCosman et al., Nature 312:768 (1984); the IL-4 signal peptide describedin EP 367,566; the type I IL-1 receptor signal peptide described in U.S.Pat. No. 4,968,607; and the type II IL-1 receptor signal peptidedescribed in EP 460,846.

[0057] DCSema polypeptides of the present invention, as isolated,purified or homogeneous proteins may be produced by recombinantexpression systems as described above or purified from naturallyoccurring cells. The polypeptides can be purified to substantialhomogeneity, as indicated by a single protein band upon analysis bySDS-polyacrylamide gel electrophoresis (SDS-PAGE).

[0058] One process for producing polypeptides of the present inventioncomprises culturing a host cell transformed with an expression vectorcomprising a DNA sequence that encodes the desired DCSema polypeptideunder conditions sufficient to promote expression of the DCSemapolypeptide. The DCSema polypeptide is then recovered from culturemedium or cell extracts, depending upon the expression system employed.As is known to the skilled artisan, procedures for purifying arecombinant protein will vary according to such factors as the type ofhost cells employed and whether or not the recombinant protein issecreted into the culture medium.

[0059] For example, when expression systems that secrete the recombinantprotein are employed, the culture medium first may be concentrated usinga commercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium. Alternatively, an anion exchangeresin can be employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,(e.g., silica gel having pendant methyl or other aliphatic groups) canbe employed to further purify the polypeptide. Some or all of theforegoing purification steps, in various combinations, are well knownand can be employed to provide a substantially homogeneous recombinantprotein.

[0060] It is possible to utilize an affinity column comprising thereceptor-binding domain of a DCSema of the present inventionaffinity-purify semaphorin receptor to which the DCSema binds. Such areceptor can be removed from an affinity column using conventionaltechniques, e.g., in a high salt elution buffer and then dialyzed into alower salt buffer for use or by changing pH or other componentsdepending on the affinity matrix utilized.

[0061] Recombinant protein produced in bacterial culture can be isolatedby initial disruption of the host cells, centrifugation, extraction fromcell pellets if an insoluble polypeptide, or from the supernatant fluidif a soluble polypeptide, followed by one or more concentration,salting-out, ion exchange, affinity purification or size exclusionchromatography steps. Finally, RP-HPLC can be employed for finalpurification steps. Microbial cells can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

[0062] Transformed yeast host cells are preferably employed to expressDCSemas of the present invention as a secreted polypeptide in order tosimplify purification. Secreted recombinant polypeptide from a yeasthost cell fermentation can be purified by methods analogous to thosedisclosed by Urdal et al. (J. Chromatog. 296:171, 1984). Urdal et al.describe two sequential, reversed-phase HPLC steps for purification ofrecombinant human IL-2 on a preparative HPLC column.

[0063] Useful fragments of the DCSema nucleic acids of the presentinvention include antisense or sense oligonucleotides comprising asingle-stranded nucleic acid sequence (either RNA or DNA) capable ofbinding to target DCSema mRNA (sense) or DCSema DNA (antisense)sequences. Antisense or sense oligonucleotides, according to the presentinvention, comprise a fragment of the coding region of DCSema cDNA. Sucha fragment generally comprises at least about 14 nucleotides, preferablyfrom about 14 to about 30 nucleotides. The ability to derive anantisense or a sense oligonucleotide, based upon a cDNA sequenceencoding a given protein is described in, for example, Stein and Cohen(Cancer Res. 48:2659, 1988) and van der Krol et al. (BioTechniques6:958, 1988).

[0064] Binding of antisense or sense oligonucleotides to target nucleicacid sequences results, in the formation of duplexes that blocktranscription or translation of the target sequence by one of severalmeans, including enhanced degradation of the duplexes, prematuretermination of transcription or translation, or by other means. Theantisense oligonucleotides thus may be used to block expression ofproteins of the present invention. Antisense or sense oligonucleotidesfurther comprise oligonucleotides having modified sugar-phosphodiesterbackbones (or other sugar linkages, such as those described inWO91/06629) and wherein such sugar linkages are resistant to endogenousnucleases. Such oligonucleotides with resistant sugar linkages arestable in vivo (i.e., capable of resisting enzymatic degradation) butretain sequence specificity to be able to bind to target nucleotidesequences. Other examples of sense or antisense oligonucleotides includethose oligonucleotides which are covalently linked to organic moieties,such as those described in WO 90/10448, and other moieties thatincreases affinity of the oligonucleotide for a target nucleic acidsequence, such as poly-(L-lysine). Further still, intercalating agents,such as ellipticine, and alkylating agents or metal complexes may beattached to sense or antisense oligonucleotides to modify bindingspecificities of the antisense or sense oligonucleotide for the targetnucleotide sequence.

[0065] Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. Antisense or sense oligonucleotides are preferably introducedinto a cell containing the target nucleic acid sequence by insertion ofthe antisense or sense oligonucleotide into a suitable retroviralvector, then contacting the cell with the retrovirus vector containingthe inserted sequence, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see PCT Application US90/02656).

[0066] Sense or antisense oligonucleotides also may be introduced into acell containing the target nucleotide sequence by formation of aconjugate with a ligand binding molecule, as described in WO 91/04753.Suitable ligand binding molecules include, but are not limited to, cellsurface receptors, growth factors, other cytokines, or other ligandsthat bind to cell surface receptors. Preferably, conjugation of theligand binding molecule does not substantially interfere with theability of the ligand binding molecule to bind to its correspondingmolecule or receptor, or block entry of the sense or antisenseoligonucleotide or its conjugated version into the cell.

[0067] Alternatively, a sense or an antisense oligonucleotide may beintroduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. The sense or antisense oligonucleotide-lipid complex ispreferably dissociated within the cell by an endogenous lipase.

[0068] In addition to the above, the following examples are provided toillustrate particular embodiments and not to limit the scope of theinvention.

EXAMPLE 1 Isolating a Human Semaphorin Homologue Designated DCSema

[0069] Using sequence alignment techniques and comparative sequenceanalysis of ESTs, a human homologue of AHV Sema semaphorin wasidentified as follows. The nucleotide sequence of viral A39R was used ina Unigene comparative sequence search. This search resulted in theidentification of EST #151129 (accession # [H029021], deposited June 20,1995. EST 151129 is a partial sequence having neither an initiating ATGnor a termination codon. EST 151129 has sequence homology to A39R andAHVsema.

[0070] EST 151129 was utilized to isolate and identify the native humanDCSema as follows:

[0071] A tissue source of EST 151129 was identified using phage libraryscreening methods and PCR primers based upon this EST sequence.Oligonucleotide PCR primers having the following nucleotide sequenceswere synthesized: 5′-TGCTGGAACCTGGTGAATGG-3′ (SEQ ID NO:3)5′-AGTGGAACAATGGCGTCTTC-3′ (SEQ ID NO:4)

[0072] PCR isolation and amplification methodologies were carried outusing a panel of human tissue cDNA phase libraries as templates for thePCR reactions.

[0073] Two of the phage libraries, human foreskin fibroblast and dermalfibroblast, were chosen for additional analysis. The libraries wereplated according to established procedures. A radiolabeled probe wasgenerated by incorporating ³²P-dCTP in the amplification of a PCRproduct using the EST 151129 as a template. The PCR reaction mixtureincluded 10 ng of EST151129 plasmid DNA, 50 pmoles of each PCRoligonucleotide primer identified below and specific for the 5′ end ofEST 151129, 1× Amplitaq buffer (Perkin-Elmer Cetus), 1.25 mM of dATP,dGTP, dTTP, 0.001 mM dCTP (Pharmacia,), 150 μCi ³²P-dCTP, 0.5 μlAmplitaq taq polymerase (Perkin-Elmer Cetus,) in a 100 μl final reactionvolume. The PCR reaction cycles included one cycle at 94° C. for 5 min;twenty-five cycles at 94° C. for 1 min, 72° C. for 2 min and one cycleat 72° C. for 5 min using a Robocycler Gradient 40 (Stratagene, LaJolla. CA).

[0074] The primers used were as follows:5′-TCCGCCCAGGGCCACCTAAGGAGCGGA-3′ (SEQ ID NO:7)5′-TGTGCGGCTCAGTCTGGCCAAAGTCCA-3′ (SEQ ID NO:8)

[0075] Approximately 5×10⁵ cpm/mL of purified probe was used tohybridize with the human dermal fibroblast library on nylon membranefilters in the same manner described in Example 5 for probing humanforeskin fibroblast and human dermal fibroblast libraries.

[0076] A cDNA that overlapped with the 5′ end of EST 151129 wasisolated. Using sequence alignments it was determined that EST 151129sequence included nucleotides 115-1536, encoding amino acids 39-512 ofthe full length peptide. The isolated cDNA provided nucleotides 1-114and additional upstream non-coding nucleotides. These added nucleotidesencode amino acids 1-38 of the full length human semaphorin, amino acid1 being the initiating methionine.

[0077] In order to isolate cDNA overlapping the 3′ end of EST 151129,another radiolabeled PCR probe was generated as described above butusing PCR oligonucleotide primers specific for the 3′ end of EST 151129:5′-AGCCAGGTGCCCCTGGACCT-3′ (SEQ ID NO:9) 5′-CTCGAGGCCAAGAATTCGGC-3′ (SEQID NO:10)

[0078] Approximately 5×10⁵ cpm/mL of purified probe was used tohybridize with the human foreskin fibroblast library on nylon membranefilters following the hybridization and washing protocol describedabove. Three independent cDNA clones were isolated which overlapped withthe 3′ end of EST151129 and extended the 3′ end of EST 151129 fromnucleotide 1537 to 2001. The isolated clones contained additionaldownstream non-coding nucleotides. Nucleotides 1537-2001 translate intoamino acids 513-666 of the full length peptide plus a stop codon. Thefull cDNA sequence of the human DCSema homologue is provided in SEQ IDNO: 1. The encoded amino acid sequence is shown in SEQ ID NO:2.

EXAMPLE 2 Northern Blot Analysis for Tissue Expressing the HumanHomologue

[0079] Radiolabeled probes derived from SEQ ID NO:1 were used inNorthern blot analyses of different human tissues to identify the tissuedistribution of the human homologue of AHV Sema, designated DCSema.

[0080] Human poly A+ multiple tissue northern blots were purchased formClontech Laboratories, Palo Alto, Calif. (Cat. #s 7760-1,7759-1, 7756-1,757-1). The northern blot filters were prehybridized, probed, and washedaccording to manufacturer's instructions. The probe was an antisenseriboprobe specific for EST 151129, an EST discovered using the viral AHVSema sequence in sequence alignment analyses and comparative analysismethods. The template for the riboprobe was generated using PCRtechniques and oligonucleotide primers that were designed to spannucleotides 613 to 978 of EST 151129(HO2902/H). The primers sequenceswere as follows: 5′ Primer: TCTACTACTT CTTCC (SEQ ID NO:5) 3′ PrimerGGAATCCTAA TACGACTCAC TATAGGGAGG CGGGTTGGGA AGGC (SEQ ID NO:6)

[0081] The underlined portion of the downstream primer is a T7 site.

[0082] The riboprobe was generated using Ambion's MAXIscript SP6/T7 kitby combining 3 μL of RNAse free water, 2 μL 10× transcription buffer, 1μL each of 10 mM dATP/dCTP/dGTP, 5 μL 5′/3′ EST 151129 PCR Product, 5 μLAmersham [α]³²P]UTP 10 mCi/mL, and 2 μL T7 RNA polymerase at roomtemperature. The combination was microfuged, spun briefly, and incubatedat 37° C. for 30 minutes. Then 1 μL DNAse was added to the mixture andallowed to react for 15 minutes at 37° C. The reaction product waspassed through two column volumes of G-25 (Boehringer). One microliter(1 μL) of the riboprobe was counted in a scintillation counter for 1minute to determine cpm/μL.

[0083] After probing the northern blots, they were washed once for 30minutes with 2×SSC, 0.05% SDS at 63° C. and three times for 30 minuteswith 0.1×SSC, 0.1% SDS and then exposed to x-ray film. The developedfilm indicated that the human DCSema homologue is found in placenta,brain, spinal cord, testes and spleen. Weak hybridization signals wereobserved in skeletal muscle, lymph node, ovary, and bone marrow.

EXAMPLE 3 Preparing a DC-Sema/Fc Fusion Protein

[0084] This example describes preparing a DC-Sema/Fc DNA construct andsubsequently expressing a DC-Sema/immunoglobulin Fc fusion proteinreferred to as DC-Sema/Fc. DNA encoding DC-Sema/Fc included a nucleotidesequence that encodes a murine IL-7 leader peptide, a FLAG™ octapeptide(described in U.S. Pat. No. 5,011,912), an Fc region of animmunoglobulin mutated to minimize binding to Fc receptor (described byBaum et al. Cir. Sh. 44:30, 1994), a flexible linker sequence and DNAencoding amino acids 45-666 of SEQ ID NO:2. An expression vectorcontaining the leader sequence, FLAG, mutated hu IgG Fc and flexiblelinker was prepared using conventional enzyme cutting and ligationtechniques. The resulting vector was then restricted with Spe1 and Not1.The DC-Sema was inserted 5′ to 3′ after the flexible linker in a two-wayligation described below.

[0085] To prepare the DC-Sema DNA, three primer pairs were designed andused to amplify three DNA fragments. Two of the fragments were amplifiedfrom a human foreskin fibroblast library phage clone containing the 3′portion of the DCSema cDNA and one fragment was EST DNA (ResearchGenetics Imageclone ID #151129). The three amplified DNA fragments werecombined in a PCR SOEing reaction to generate a DNA fragment encodingthe entire peptide of DC-Sema (Horton et al., Biotechniques 8:528,1990). In the final fragment, the upstream oligonucleotide primerintroduced a Spe1 site upstream of amino acid 45 of the DC-sema peptide.A downstream oligonucleotide primer introduced a Not1 site justdownstream of the termination codon after amino acid 666.

[0086] The PCR fragment was then ligated into an expression vector(pDC409) containing the leader sequence. Flag® sequence, mutated humanIgG Fc and a flexible linker region in a two-way ligation. The resultingDNA construct was transfected into the monkey kidney cell linesCV-1/EBNA. After 7 days of culture in medium containing 0.5% lowimmunoglobulin bovine serum, a solution of 0.2% azide was added to thesupernatant and the supernatant was filtered through a 0.22 μm filter.Then approximately 1 L of culture supernatant was passed through aBioCad Protein A HPLC protein purification system using a 4.6×100 mmProtein A column (POROS 20A from PerSeptive Biosystems) at 10 mL/min.The Protein A column binds the Fc Portion of the fusion protein in thesupernatant, immobilizing the fusion protein and allowing othercomponents of the supernatant to pass through the column. The column waswashed with 30 mL of PBS solution and bound fusion protein was elutedfrom the HPLC column with citric acid adjusted to pH 3.0. Elutedpurified fusion protein was neutralized as it eluted using 1M HEPESsolution at pH 7.4.

EXAMPLE 4 Preparing DC-Sema/polyHIS Fusion Protein

[0087] This example describes preparing a DC-Sema/polyHIS DNA constructand subsequently expressing a DCSema/poly histidine tagged proteinreferred to as DC-sema/polyHIS fusion protein. DNA encodingDC-sema/polyHIS comprises sequences encoding the DC-sema gene from aminoacid 1 to amino acid 666 (no stop codon), followed by a factor Xacleavage site, a FLAG® octapeptide, a string of six histidine residuesand a stop codon. An expression vector containing the factor Xa cleavagesite, flag, and polyHIS sequence was prepared using conventional enzymecutting and fragment ligation techniques. The resulting vector was thenrestricted with Sal1 and SnaB 1. The DCSema sequence was inserted 5′ to3′ before the factor Xa cleavage site in a two-way ligation describedbelow.

[0088] To prepare the DCSema DNA, three primer pairs were designed andused to amplify three DNA fragments. Two of the fragments were fromphage DNA and one fragment was EST DNA (Research Genetics Imageclone ID#151129). The three amplified DNA fragments were combined in a PCRSOEing reaction to generate a DNA fragment encoding the entire peptideof DCSema. This final DNA included a Sal1 site upstream of amino acid 1of the DCSema peptide and a SnaB1 site downstream of amino acid 666.

[0089] The PCR product was then ligated into an expression vector pDC409containing the factor Xa cleavage site, flag, and polyHIS sequence in atwo-way ligation. The resultant DNA construct (DCSema/polyHIS) wastransiently transfected into the monkey cell line COS-1 (ATCC CRL-1650).Following a 7 day culture in medium containing 0.5% low immunoglobulinbovine serum, cell supernatants were harvested and a solution of 0.2%sodium azide was added to the supernatants. The supernatants werefiltered through a 0.22 μm filter, concentrated 10 fold with a prepscale concentrator (Millipore: Bedford, Mass.) and purified on a BioCadHPLC protein purification equipped with a Nickel NTA Superflow self packresin column (Qiagen, Santa Clarita, Calif.). After the supernatantpassed through the column, the column was washed with Buffer A (20 mMNaPO4, pH7.4; 300 mMNaCl; 50 mM Imidazole). Bound protein was theneluted from the column using a gradient elution techniques. Fractionscontaining protein were collected and analyzed on a 4-20% SDS-PAGEreducing gel. Peaks containing the fusion protein were pooled,concentrated 2 fold, and then dialyzed in PBS. The resultingDCSema/polyHis fusion protein was then filtered through a 0.22 μmsterile filter and recovered.

EXAMPLE 5 Screening Cell Lines for Binding to DCSema

[0090] The DC-Sema/Fc fusion protein prepared as described in Example 3was used to screen cell lines for binding using quantitative bindingstudies according to standard flow cytometry methodologies. For eachcell line screened, the procedure involved incubating approximately100,000 of the cells blocked with 2% FCS (fetal calf serum), 5% normalgoat serum and 5% rabbit serum in PBS for 1 hour. Then the blocked cellswere incubated with 5 μg/mL of DC-Sema/Fc fusion protein in 2% FCS, 5%goat serum and 5% rabbit serum in PBS. Following the incubation thesample was washed 2 times with FACS buffer (2% FCS in PBS) and thentreated with mouse anti human Fc/biotin (purchased from JacksonResearch) and SAPE (streptavidin-phycoerythrin purchased from MolecularProbes). This treatment causes the antihuman Fc/biotin to bind to anybound DC-Sema/Fc and the SAPE to bind to the anti human Fc/biotinresulting in a fluorescent identifying label on DC-Sema/Fc which isbound to cells. The cells were analyzed for any bound protein usingfluorescent detection flow cytometry. Table I details the results of theflow cytometry studies. +indicates that binding was detected between thecell surface and DC-Sema. −indicates that no binding was detectedbetween the cell surface and A39R. TABLE I Cell Line DC-Sema BindingResult CB23 (Human Cord Blood B Cell Line) + MP-1 (Human B CellLymphoma) + PB B (Human Peripheral Blood B Cells) + U937 (HumanMonocyte-Type Cell) + W126 (Human Lung Epithelium) + RAJI (Burkitt'sLymphoma) + Primary Human Monocytes + THP-1 (Human Promonocytes) +

EXAMPLE 6 Monoclonal Antibodies to DC-Sema

[0091] This example illustrates a method for preparing antibodies toDC-Sema. Purified DC-Sema/Fc is prepared as described in Example 3above. The purified protein is used to generate antibodies againstDC-Sema as described in U.S. Pat. No. 4,411,993. Briefly, mice areimmunized at 0, 2 and 6 weeks with 10 μg with DCSema/Fc. The primaryimmunization is prepared with TITERMAX adjuvant, from Vaxcell, Inc., andsubsequent immunizations are prepared with incomplete Freund's adjuvant(IFA). At 11 weeks, the mice are IV boosted with 3-4 μg DCSema/Fc inPBS. Three days after the IV boost, splenocytes are harvested and fusedwith an Ag8.653 myeloma fusion partner using 50% aqueous PEG 1500solution. Hybridoma supernatants are screened for DC-Sema antibodies bydot blot assay against DCSema/FC and an irrelevant Fc protein.

EXAMPLE 7 Cytokine Induction from Freshly Isolated Human Monocytes

[0092] Freshly isolated human monocytes were purified by first diluting1:1 peripheral blood from healthy donors in low endotoxin PBS at pH 7.4and room temperature. Then 35 mLs of the diluted blood was layered over15 mLs of Isolymph (Gallard and Schlesinger Industries, Inc; CarlePlace, N.Y.) and centrifuged at 2200 rpm for 25 minutes at roomtemperature. The plasma layers was reserved. The PBMC layer washarvested and washed three times to remove the Isolymph. The washedPBMC's were resuspended in X-Vivo 15 serum free media (BioWhittaker,Walkersville, Md.) and added to T175 flasks. The flasks had beenpreviously coated with 2% Gelatin (Sigma, St. Louis, Mo.) andpre-treated for 30 minutes with the reserved plasma layer. The PBMC'swere allowed to adhere for 90 minutes at 37° C., 5% CO₂ and then rinsedthree times gently with 10 mL washes of low endotoxin PBS. Adheredmonocytes were harvested by incubating the cells in Enzyme FreeDissociation Buffer (Gibco, BRL) and washing the cells multiple times inPBS. Monocytes were centrifuged at 2500 rpm for 5 minutes, counted, andset up in 24 well dishes at 5×10⁵ cells/well in 1 mL. The cultures were95% pure.

[0093] Purified monocytes were cultured for 7-9 days in the presence of20 ng/mL GM-CSF and 100 ng/mL IL-4 in order to allow cells todifferentiate to a more dendritic cell-like phenotype. On day 7-9,cultures were treated with 1 μg/mL DCSema/Fc fusion protein (see Example3) or a control Fc protein, and the next day cells and supernatants wereharvested for analyses.

[0094] The monocyte supernatants were examined for the presence ofproinflammatory cytokines. In all donors tested, IL-6 and IL-8 wasinduced by DCSema protein. Heat inactivated DCSema and control proteinsdid not induce IL-6 or IL-8. Additionally, cytokine production wasblocked by the inclusion of a mAb directed against DCSema.

[0095] The results of this experiment demonstrate that DCSema, orhomologues of this protein, by interact with its receptor, can inducecytokine production by freshly isolated monocytes.

EXAMPLE 8 Monocyte Aggregation Studies

[0096] In order to examine human monocyte response to the interaction ofa DCSema to its receptor on monocytes, monocytes were purified asdescribed in Example 7 and DC-Sema/Fc fusion protein was prepared asdescribed in Example 3. After incubating the DCSema/Fc fusion proteinand purified, cultured monocytes for 20 hours, monocyte aggregation wasobserved.

[0097] This work confirms that the receptor for the DCSema of thepresent invention is expressed on monocytes and that the interactionbetween DCSema and it receptor results in monocyte aggregation. Similarto B cells, monocyte aggregation is indicative of their activation.

1 10 1 2001 DNA Homo sapiens CDS (1)..(2001) 1 atg acg cct cct ccg cccgga cgt gcc gcc ccc agc gca ccg cgc gcc 48 Met Thr Pro Pro Pro Pro GlyArg Ala Ala Pro Ser Ala Pro Arg Ala 1 5 10 15 cgc gtc cct ggc ccg ccggct cgg ttg ggg ctt ccg ctg cgg ctg cgg 96 Arg Val Pro Gly Pro Pro AlaArg Leu Gly Leu Pro Leu Arg Leu Arg 20 25 30 ctg ctg ctg ctg ctt tgg gcggcc gcc gcc tcc gcc cag ggc cac cta 144 Leu Leu Leu Leu Leu Trp Ala AlaAla Ala Ser Ala Gln Gly His Leu 35 40 45 agg agc gga ccc cgc atc ttc gccgtc tgg aaa ggc cat gta ggg cag 192 Arg Ser Gly Pro Arg Ile Phe Ala ValTrp Lys Gly His Val Gly Gln 50 55 60 gac cgg gtg gac ttt ggc cag act gagccg cac acg gtg ctt ttc cac 240 Asp Arg Val Asp Phe Gly Gln Thr Glu ProHis Thr Val Leu Phe His 65 70 75 80 gag cca ggc agc tcc tct gtg tgg gtggga gga cgt ggc aag gtc tac 288 Glu Pro Gly Ser Ser Ser Val Trp Val GlyGly Arg Gly Lys Val Tyr 85 90 95 ctc ttt gac ttc ccc gag ggc aag aac gcatct gtg cgc acg gtg aat 336 Leu Phe Asp Phe Pro Glu Gly Lys Asn Ala SerVal Arg Thr Val Asn 100 105 110 atc ggc tcc aca aag ggg tcc tgt ctg gataag cgg gac tgc gag aac 384 Ile Gly Ser Thr Lys Gly Ser Cys Leu Asp LysArg Asp Cys Glu Asn 115 120 125 tac atc act ctc ctg gag agg cgg agt gagggg ctg ctg gcc tgt ggc 432 Tyr Ile Thr Leu Leu Glu Arg Arg Ser Glu GlyLeu Leu Ala Cys Gly 130 135 140 acc aac gcc cgg cac ccc agc tgc tgg aacctg gtg aat ggc act gtg 480 Thr Asn Ala Arg His Pro Ser Cys Trp Asn LeuVal Asn Gly Thr Val 145 150 155 160 gtg cca ctt ggc gag atg aga ggc tacgcc ccc ttc agc ccg gac gag 528 Val Pro Leu Gly Glu Met Arg Gly Tyr AlaPro Phe Ser Pro Asp Glu 165 170 175 aac tcc ctg gtt ctg ttt gaa ggg gacgag gtg tat tcc acc atc cgg 576 Asn Ser Leu Val Leu Phe Glu Gly Asp GluVal Tyr Ser Thr Ile Arg 180 185 190 aag cag gaa tac aat ggg aag atc cctcgg ttc cgc cgc atc cgg ggc 624 Lys Gln Glu Tyr Asn Gly Lys Ile Pro ArgPhe Arg Arg Ile Arg Gly 195 200 205 gag agt gag ctg tac acc agt gat actgtc atg cag aac cca cag ttc 672 Glu Ser Glu Leu Tyr Thr Ser Asp Thr ValMet Gln Asn Pro Gln Phe 210 215 220 atc aaa gcc acc atc gtg cac caa gaccag gct tac gat gac aag atc 720 Ile Lys Ala Thr Ile Val His Gln Asp GlnAla Tyr Asp Asp Lys Ile 225 230 235 240 tac tac ttc ttc cga gag gac aatcct gac aag aat cct gag gct cct 768 Tyr Tyr Phe Phe Arg Glu Asp Asn ProAsp Lys Asn Pro Glu Ala Pro 245 250 255 ctc aat gtg tcc cgt gtg gcc cagttg tgc agg ggg gac cag ggt ggg 816 Leu Asn Val Ser Arg Val Ala Gln LeuCys Arg Gly Asp Gln Gly Gly 260 265 270 gaa agt tca ctg tca gtc tcc aagtgg aac act ttt ctg aaa gcc atg 864 Glu Ser Ser Leu Ser Val Ser Lys TrpAsn Thr Phe Leu Lys Ala Met 275 280 285 ctg gta tgc agt gat gct gcc accaac aag aac ttc aac agg ctg caa 912 Leu Val Cys Ser Asp Ala Ala Thr AsnLys Asn Phe Asn Arg Leu Gln 290 295 300 gac gtc ttc ctg ctc cct gac cccagc ggc cag tgg agg gac acc agg 960 Asp Val Phe Leu Leu Pro Asp Pro SerGly Gln Trp Arg Asp Thr Arg 305 310 315 320 gtc tat ggt gtt ttc tcc aacccc tgg aac tac tca gcc gtc tgt gtg 1008 Val Tyr Gly Val Phe Ser Asn ProTrp Asn Tyr Ser Ala Val Cys Val 325 330 335 tat tcc ctc ggt gac att gacaag gtc ttc cgt acc tcc tca ctc aag 1056 Tyr Ser Leu Gly Asp Ile Asp LysVal Phe Arg Thr Ser Ser Leu Lys 340 345 350 ggc tac cac tca agc ctt cccaac ccg cgg cct ggc aag tgc ctc cca 1104 Gly Tyr His Ser Ser Leu Pro AsnPro Arg Pro Gly Lys Cys Leu Pro 355 360 365 gac cag cag ccg ata ccc acagag acc ttc cag gtg gct gac cgt cac 1152 Asp Gln Gln Pro Ile Pro Thr GluThr Phe Gln Val Ala Asp Arg His 370 375 380 cca gag gtg gcg cag agg gtggag ccc atg ggg cct ctg aag acg cca 1200 Pro Glu Val Ala Gln Arg Val GluPro Met Gly Pro Leu Lys Thr Pro 385 390 395 400 ttg ttc cac tct aaa taccac tac cag aaa gtg gcc gtt cac cgc atg 1248 Leu Phe His Ser Lys Tyr HisTyr Gln Lys Val Ala Val His Arg Met 405 410 415 caa gcc agc cac ggg gagacc ttt cat gtg ctt tac cta act aca gac 1296 Gln Ala Ser His Gly Glu ThrPhe His Val Leu Tyr Leu Thr Thr Asp 420 425 430 agg ggc act atc cac aaggtg gtg gaa ccg ggg gag cag gag cac agc 1344 Arg Gly Thr Ile His Lys ValVal Glu Pro Gly Glu Gln Glu His Ser 435 440 445 ttc gcc ttc aac atc atggag atc cag ccc ttc cgc cgc gcg gct gcc 1392 Phe Ala Phe Asn Ile Met GluIle Gln Pro Phe Arg Arg Ala Ala Ala 450 455 460 atc cag acc atg tcg ctggat gct gag cgg agg aag ctg tat gtg agc 1440 Ile Gln Thr Met Ser Leu AspAla Glu Arg Arg Lys Leu Tyr Val Ser 465 470 475 480 tcc cag tgg gag gtgagc cag gtg ccc ctg gac ctg tgt gag gtc tat 1488 Ser Gln Trp Glu Val SerGln Val Pro Leu Asp Leu Cys Glu Val Tyr 485 490 495 ggc ggg ggc tgc cacggt tgc ctc atg tcc cga gac ccc tac tgc ggc 1536 Gly Gly Gly Cys His GlyCys Leu Met Ser Arg Asp Pro Tyr Cys Gly 500 505 510 tgg gac cag ggc cgctgc atc tcc atc tac agc tcc gaa cgg tca gtg 1584 Trp Asp Gln Gly Arg CysIle Ser Ile Tyr Ser Ser Glu Arg Ser Val 515 520 525 ctg caa tcc att aatcca gcc gag cca cac aag gag tgt ccc aac ccc 1632 Leu Gln Ser Ile Asn ProAla Glu Pro His Lys Glu Cys Pro Asn Pro 530 535 540 aaa cca gac aag gcccca ctg cag aag gtt tcc ctg gcc cca aac tct 1680 Lys Pro Asp Lys Ala ProLeu Gln Lys Val Ser Leu Ala Pro Asn Ser 545 550 555 560 cgc tac tac ctgagc tgc ccc atg gaa tcc cgc cac gcc acc tac tca 1728 Arg Tyr Tyr Leu SerCys Pro Met Glu Ser Arg His Ala Thr Tyr Ser 565 570 575 tgg cgc cac aaggag aac gtg gag cag agc tgc gaa cct ggt cac cag 1776 Trp Arg His Lys GluAsn Val Glu Gln Ser Cys Glu Pro Gly His Gln 580 585 590 agc ccc aac tgcatc ctg ttc atc gag aac ctc acg gcg cag cag tac 1824 Ser Pro Asn Cys IleLeu Phe Ile Glu Asn Leu Thr Ala Gln Gln Tyr 595 600 605 ggc cac tac ttctgc gag gcc cag gag ggc tcc tac ttc cgc gag gct 1872 Gly His Tyr Phe CysGlu Ala Gln Glu Gly Ser Tyr Phe Arg Glu Ala 610 615 620 cag cac tgg cagctg ctg ccc gag gac ggc atc atg gcc gag cac ctg 1920 Gln His Trp Gln LeuLeu Pro Glu Asp Gly Ile Met Ala Glu His Leu 625 630 635 640 ctg ggt catgcc tgt gcc ctg gct gcc tcc ctc tgg ctg ggg gtg ctg 1968 Leu Gly His AlaCys Ala Leu Ala Ala Ser Leu Trp Leu Gly Val Leu 645 650 655 ccc aca ctcact ctt ggc ttg ctg gtc cac tag 2001 Pro Thr Leu Thr Leu Gly Leu Leu ValHis 660 665 2 666 PRT Homo sapiens 2 Met Thr Pro Pro Pro Pro Gly Arg AlaAla Pro Ser Ala Pro Arg Ala 1 5 10 15 Arg Val Pro Gly Pro Pro Ala ArgLeu Gly Leu Pro Leu Arg Leu Arg 20 25 30 Leu Leu Leu Leu Leu Trp Ala AlaAla Ala Ser Ala Gln Gly His Leu 35 40 45 Arg Ser Gly Pro Arg Ile Phe AlaVal Trp Lys Gly His Val Gly Gln 50 55 60 Asp Arg Val Asp Phe Gly Gln ThrGlu Pro His Thr Val Leu Phe His 65 70 75 80 Glu Pro Gly Ser Ser Ser ValTrp Val Gly Gly Arg Gly Lys Val Tyr 85 90 95 Leu Phe Asp Phe Pro Glu GlyLys Asn Ala Ser Val Arg Thr Val Asn 100 105 110 Ile Gly Ser Thr Lys GlySer Cys Leu Asp Lys Arg Asp Cys Glu Asn 115 120 125 Tyr Ile Thr Leu LeuGlu Arg Arg Ser Glu Gly Leu Leu Ala Cys Gly 130 135 140 Thr Asn Ala ArgHis Pro Ser Cys Trp Asn Leu Val Asn Gly Thr Val 145 150 155 160 Val ProLeu Gly Glu Met Arg Gly Tyr Ala Pro Phe Ser Pro Asp Glu 165 170 175 AsnSer Leu Val Leu Phe Glu Gly Asp Glu Val Tyr Ser Thr Ile Arg 180 185 190Lys Gln Glu Tyr Asn Gly Lys Ile Pro Arg Phe Arg Arg Ile Arg Gly 195 200205 Glu Ser Glu Leu Tyr Thr Ser Asp Thr Val Met Gln Asn Pro Gln Phe 210215 220 Ile Lys Ala Thr Ile Val His Gln Asp Gln Ala Tyr Asp Asp Lys Ile225 230 235 240 Tyr Tyr Phe Phe Arg Glu Asp Asn Pro Asp Lys Asn Pro GluAla Pro 245 250 255 Leu Asn Val Ser Arg Val Ala Gln Leu Cys Arg Gly AspGln Gly Gly 260 265 270 Glu Ser Ser Leu Ser Val Ser Lys Trp Asn Thr PheLeu Lys Ala Met 275 280 285 Leu Val Cys Ser Asp Ala Ala Thr Asn Lys AsnPhe Asn Arg Leu Gln 290 295 300 Asp Val Phe Leu Leu Pro Asp Pro Ser GlyGln Trp Arg Asp Thr Arg 305 310 315 320 Val Tyr Gly Val Phe Ser Asn ProTrp Asn Tyr Ser Ala Val Cys Val 325 330 335 Tyr Ser Leu Gly Asp Ile AspLys Val Phe Arg Thr Ser Ser Leu Lys 340 345 350 Gly Tyr His Ser Ser LeuPro Asn Pro Arg Pro Gly Lys Cys Leu Pro 355 360 365 Asp Gln Gln Pro IlePro Thr Glu Thr Phe Gln Val Ala Asp Arg His 370 375 380 Pro Glu Val AlaGln Arg Val Glu Pro Met Gly Pro Leu Lys Thr Pro 385 390 395 400 Leu PheHis Ser Lys Tyr His Tyr Gln Lys Val Ala Val His Arg Met 405 410 415 GlnAla Ser His Gly Glu Thr Phe His Val Leu Tyr Leu Thr Thr Asp 420 425 430Arg Gly Thr Ile His Lys Val Val Glu Pro Gly Glu Gln Glu His Ser 435 440445 Phe Ala Phe Asn Ile Met Glu Ile Gln Pro Phe Arg Arg Ala Ala Ala 450455 460 Ile Gln Thr Met Ser Leu Asp Ala Glu Arg Arg Lys Leu Tyr Val Ser465 470 475 480 Ser Gln Trp Glu Val Ser Gln Val Pro Leu Asp Leu Cys GluVal Tyr 485 490 495 Gly Gly Gly Cys His Gly Cys Leu Met Ser Arg Asp ProTyr Cys Gly 500 505 510 Trp Asp Gln Gly Arg Cys Ile Ser Ile Tyr Ser SerGlu Arg Ser Val 515 520 525 Leu Gln Ser Ile Asn Pro Ala Glu Pro His LysGlu Cys Pro Asn Pro 530 535 540 Lys Pro Asp Lys Ala Pro Leu Gln Lys ValSer Leu Ala Pro Asn Ser 545 550 555 560 Arg Tyr Tyr Leu Ser Cys Pro MetGlu Ser Arg His Ala Thr Tyr Ser 565 570 575 Trp Arg His Lys Glu Asn ValGlu Gln Ser Cys Glu Pro Gly His Gln 580 585 590 Ser Pro Asn Cys Ile LeuPhe Ile Glu Asn Leu Thr Ala Gln Gln Tyr 595 600 605 Gly His Tyr Phe CysGlu Ala Gln Glu Gly Ser Tyr Phe Arg Glu Ala 610 615 620 Gln His Trp GlnLeu Leu Pro Glu Asp Gly Ile Met Ala Glu His Leu 625 630 635 640 Leu GlyHis Ala Cys Ala Leu Ala Ala Ser Leu Trp Leu Gly Val Leu 645 650 655 ProThr Leu Thr Leu Gly Leu Leu Val His 660 665 3 20 DNA Artificial SequencePRIMER 3 tgctggaacc tggtgaatgg 20 4 20 DNA Artificial Sequence PRIMER 4agtggaacaa tggcgtcttc 20 5 15 DNA Artificial Sequence PRIMER 5tctactactt cttcc 15 6 44 DNA Artificial Sequence PRIMER 6 ggaatcctaatacgactcac tatagggagg cgggttggga aggc 44 7 27 DNA Artificial SequencePRIMER 7 tccgcccagg gccacctaag gagcgga 27 8 27 DNA Artificial SequencePRIMER 8 tgtgcggctc agtctggcca aagtcca 27 9 20 DNA Artificial SequencePRIMER 9 agccaggtgc ccctggacct 20 10 20 DNA Artificial Sequence PRIMER10 ctcgaggcca agaattcggc 20

What is claimed is:
 1. A semaphorin polypeptide comprising an amino acidsequence that is at least 80% identical to the amino acid sequence ofSEQ ID NO:2, the semaphorin polypeptide capable of binding at least onesemaphorin receptor.
 2. An isolated semaphorin polypeptide encoded byDNA sequences selected from the group consisting of: (a) nucleotidesx₁—2001 of SEQ ID NO:1, wherein x₁ is nucleotide 1 or 135; and (b) DNAsequences that hybridize under moderately stringent conditions to theDNA of (a); and which DNA sequences encode a polypeptide that bindssemaphorin receptors.
 3. A semaphorin polypeptide comprising an aminoacid sequence that is at least 80% identical to an amino acid sequenceselected from the group consisting of: (a) amino acids x₁ to 666 of SEQID NO: 2, wherein x₁ is amino acid 1 or
 45. a fragment of the sequenceof (a), wherein the fragment is capable of binding a semaphorinreceptor.
 4. A semaphorin polypeptide comprising an amino acid sequenceselected from the group consisting of: (a) amino acids x₁ to 666 of SEQID NO: 2, wherein x₁ is amino acid 1 or 45, and (b) a fragment of thesequence of (a), wherein the fragment is capable of binding a semaphorinreceptor.
 5. An isolated DNA encoding a semaphorin polypeptide, the DNAselected from the group consisting of: (a) DNA capable of hybridizingunder moderately stringent conditions to a nucleotide sequenceconsisting essentially of SEQ ID NO: 1; and (b) DNA capable ofhybridizing under moderately stringent conditions to DNA complementaryto the sequence of (a).
 6. An isolated DNA encoding a semaphorinpolypeptide, the DNA selected from the group consisting of: (a)nucleotides x₁—2001 of SEQ ID NO:1, wherein x₁ is nucleotide 1 or 135;and (b) DNA sequences that hybridize under moderately stringentconditions to the cDNA of (a); and which DNA sequences encode apolypeptide that binds semaphorin receptor; and (c) DNA sequences that,due to the degeneracy of the genetic code, encode semaphorinpolypeptides having the amino acid sequence of the polypeptides encodedby the DNA sequences of (a) or (b).
 7. An isolated DNA encoding ansemaphorin polypeptide wherein the polypeptide comprises an amino acidsequence that is at least 80% identical to the amino acid sequence ofSEDQ ID NO:2.
 8. The isolated DNA of claim 7 wherein the semaphorinpolypeptide comprises the amino acid sequence of SEQ ID NO:2.
 9. Anisolated DNA encoding a semaphorin polypeptide, wherein the polypeptidecomprises an amino acid sequence that is at least 80% identical to asequence selected from the group consisting of: (a) amino acids x₁—666of SEQ ID NO:2, wherein x₁ is amino acid 1 or 45; and (b) a fragment ofthe sequence of (a), wherein the polypeptide binds a semaphorinreceptor.
 10. A DNA of claim 9 wherein the, semaphorin polypeptidecomprises an amino acid sequence selected from the group consisting of:(a) amino acids x₁—666 of SEQ ID NO:2, wherein x₁ is amino acid 1 or 45;and (b) a fragment of (a).
 11. A DNA that is at least 80% identical toDNA that encodes an amino acid sequence selected from the groupconsisting: (a) amino acids x₁—666 of SEQ ID NO:2, wherein x₁ is aminoacid 1 or 45; and (b) a fragment of (a).
 12. A fusion protein comprisingamino acids x₁—666 where x₁ is amino acid 1 or 45 of SEQ ID NO:2
 13. Arecombinant expression vector comprising DNA of claim
 6. 14. A processfor preparing a semaphorin polypeptide, the process comprising culturinga host cell transformed with an expression vector of claim 14 underconditions that promote expression of the polypeptide, and recoveringthe polypeptide.
 15. A composition comprising a suitable diluent carrierand a polypeptide of claim
 3. 16. An antibody that is immunoreactivewith a polypeptide of claim
 2. 17. A process for treating aninflammatory disease in a mammal afflicted with the disease, the processcomprising administering an amount of semaphorin polypeptide.
 18. Amethod of separating cells having semaphorin polypeptide on the surfacethereof from a mixture of cells in suspension, comprising contacting thecells in the mixture with a contacting surface having a semaphorin, andseparating the contacting surface and the suspension.
 19. An isolatedDNA selected from the group consisting of: (a) DNA of SEQ ID NO:5; (b)DNA sequences that hybridize under moderately stringent conditions tothe cDNA of (a); and which DNA sequences encode a polypeptide the bindssemaphorin receptor; (c) DNA sequences that, due to the degeneracy ofthe genetic code, encode semaphorin polypeptides having the amino acidsequence of the polypeptides encoded by the DNA sequences of (a) or (b).20. An isolated DNA encoding a soluble semaphorin polypeptide, whereinthe soluble polypeptide comprises an amino acid sequence that is atleast 90% identical to a sequence selected from the group consisting of:(a) amino acids x₁ to 666 of SEQ ID NO:6, wherein x₁ is amino acid 1 or45; (b) a fragment of the sequence of (a), wherein the solublepolypeptide binds a semaphorin receptor.
 21. A DNA of claim 29 whereinthe soluble polypeptide comprises an amino acid sequence selected fromthe group consisting of: (a) amino acids x₁ to 666 of SEQ ID NO:6,wherein x₁ is amino acid 1 or 45; and (b) a fragment of (a).
 22. Arecombinant expression vector comprising DNA of claim
 21. 23. A processfor preparing a semaphorin polypeptide, the process comprising culturinga host cell transformed with an expression vector of claim 31 underconditions that promote expression of the polypeptide, and recoveringthe polypeptide.